Arabidopsis Membrane-anchored Ubiquitin-fold (MUB)Proteins Localize a Specific Subset of Ubiquitin-conjugating(E2) Enzymes to the Plasma Membrane*□S

Received for publication, June 25, 2010, and in revised form, February 21, 2011 Published, JBC Papers in Press, February 23, 2011, DOI 10.1074/jbc.M110.158808

Rebecca T. Dowil‡, Xiaolong Lu‡, Scott A. Saracco§, Richard D. Vierstra¶1, and Brian P. Downes‡2

From the ‡Department of Biology, Saint Louis University, St. Louis, Missouri 63103-2010, the ¶Department of Genetics, University ofWisconsin, Madison, Wisconsin 53706-1574, and §Monsanto Company, St. Louis, Missouri 63017

The covalent attachment of ubiquitin (Ub) to various intracel-lular proteins plays important roles in altering the function,localization, processing, and degradation of themodified target.Aminimal ubiquitylation pathway uses a three-enzyme cascade(E1, E2, and E3) to activate Ub and select target proteins formodification. Although diverse E3 families provide much of thetarget specificity, several factors have emerged recently thatcoordinate the subcellular localization of the ubiquitylationmachinery. Here, we show that the family of membrane-an-chored ubiquitin-fold (MUB) proteins recruits and docks spe-cific E2s to the plasma membrane. Protein interaction screenswith Arabidopsis MUBs revealed that interacting E2s are lim-ited to awell defined subgroup that is phylogenetically related tohumanUbcH5andyeastUbc4/5 families.MUBs appear to inter-act noncovalentlywith anE2 surface opposite the active site thatforms a covalent linkage with Ub. Bimolecular fluorescencecomplementation demonstrated that MUBs bind simultane-ously to the plasma membrane via a prenyl tail and to the E2 inplanta. These findings suggest that MUBs contribute subcellu-lar specificity to ubiquitylation by docking the conjugationmachinery to the plasma membrane.

Protein ubiquitylation is a reversible post-translational mod-ification regulating target protein activity, localization, anddegradation in diverse signaling pathways (1, 2). The fate of aubiquitin (Ub)3-targeted protein ultimately depends on thelength and linkage of the attached Ub molecules (3). Forinstance, Ub Lys48-linked chains typically direct target proteinsinto the Ub/26 S proteasome system (UPS) for degradation.Ubiquitylation at the plasma membrane often differs from the

canonical UPS through reversible monoubiquitylation, proteinrecycling between themembrane and endomembrane systems,the assembly of Lys63-linked chains, and terminal lysosomaltargeting (4). For example, monoubiquitylation of plasmamembrane proteins triggers their endocytosis (1, 3, 5, 6).Ub and related proteins are part of the�-grasp protein struc-

ture family that is characterized by a transverse �-helix cradledin a five-stranded �-sheet. The C-terminal diglycine of Ub isnecessary for activation by an E1 enzyme, transfer to the activesite of a Ub-conjugating enzyme (UBC; E2), and covalentattachment to a target protein guided by an E3 enzyme. Ub-related proteins should be considered either Ub-like when theyare used in a covalent protein conjugation reaction or, if not,simply Ub-fold. Functional variation in protein conjugation isattributed to a growing list of Ub-like proteins, includingSUMO (small ubiquitin-like modifier), RUB (NEDD8), UFM,and others (7). Alternatively, Ub-fold proteins, which lack a Cterminus for protein conjugation, use the �-grasp for proteininteractions that guide subcellular organization of ubiquityla-tion. For instance, RAD23 directs ubiquitylated cargoes to the26 S proteasome, and phosphatidylethanolamine-conjugatedATG8marksmembranes in developing autophagosomes (8, 9).Throughout the UPS, the Ub-fold is recognized by various

Ub-binding domains (UBDs) from the initial E1 enzyme (10) tothe Ub conjugate docking 26 S proteasome subunits RPN1,RPN10, and RPN13 (11). An Ile44-centered hydrophobic sur-face on the exterior of the Ub �-sheet is most commonly rec-ognized byUBDs. Certain E2s can also interactwith this surfacewithout encroaching on their active site. Specifically, solution-based two-dimensional NMR studies detected Ub bound non-covalently near Ser22 on human (Homo sapiens (Hs)) UbcH5c(12), which has been confirmed by activity assays (13, 14) andcrystallography (14) for HsUbcH5b. Noncovalent Ub-E2 inter-action is proposed to aid assembly of Ub-E2 polymeric com-plexes for enhanced target protein polyubiquitylation (12, 14).Ub enzyme variants (UEVs) resemble E2s but lack an active sitecysteine, thus precluding covalent conjugation to Ub-like pro-teins. Nevertheless, UEVs can diversify E2 activities (15–17). Infact, a noncovalent interaction betweenUb and theUEVMms2promotes the UEV/E2 heterodimer Mms2/Ubc13 to formLys63-linked poly-Ub chains (16). In yeast, a related noncova-lent interaction between SUMO and its E2, Ubc9, also stimu-lates poly-SUMO chain formation (18–23).Membrane-anchored Ub-fold (MUB) proteins are recent

additions to the �-grasp structure family. Compared with Ub,

* This work was supported by Saint Louis University and a Beaumont facultydevelopment grant (to B. P. D.).

This paper is dedicated to the memory of Tim Tague (September 24, 1955 toOctober 23, 2006), father of Rebecca T. Dowil.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Figs. 1– 4 and Tables I and II.

1 Supported by National Science Foundation Arabidopsis 2010 Program GrantMCB-0115870.

2 To whom correspondence should be addressed: Dept. of Biology, SaintLouis University, 3507 Laclede Ave., St. Louis, MO 63103-2010. Tel.: 314-977-3913; Fax: 314-977-3658; E-mail: bdownes1@slu.edu.

3 The abbreviations used are: Ub, ubiquitin; UPS, Ub/26 S proteasome system;UBC, Ub-conjugating enzyme; UBD, Ub-binding domain; Hs, H. sapiens;UEV, Ub enzyme variant; MUB, membrane-anchored Ub-fold; At, A. thali-ana; Co-IP, co-immunoprecipitation; BiFC, bimolecular fluorescence com-plementation; GUS, �-glucuronidase; NLS, nuclear localization signal; Y2H,yeast two-hybrid; SUMO, small ubiquitin-like modifier.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 17, pp. 14913–14921, April 29, 2011© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

APRIL 29, 2011 • VOLUME 286 • NUMBER 17 JOURNAL OF BIOLOGICAL CHEMISTRY 14913

by guest on September 22, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Arabidopsis thaliana (At) MUB1 (Protein Data Bank code1SE9) and HsMUB (previously known as UBL3; code 2GOW)show strong three-dimensional similarity in the �-grasp, butlongerNandC termini and extended loops createMUB-uniquesurfaces (24–26). MUBs are distinguished from other Ub-foldproteins by a C-terminal CAAX box that is modified throughprotein prenylation with a hydrophobic membrane anchor.Prenylation andmembrane localization preclude attachment ofMUBs to target proteins. MUBs from nematodes, insects, fish,and mammals, including humans, are prenylated in vitro (26).In planta, AtMUB membrane localization is prenylation-de-pendent, where mutation of the prenyl attachment CAAX cys-teine to a serine (SAAX) prevents processing and causesaccumulation of non-membrane-localized MUB. Likewise,Arabidopsis prenyltransferase mutants block membrane local-ization of endogenous AtMUB1 (26). The six MUB genes ofArabidopsis are divided by sequence homology into three sub-groups, MUB1/2, MUB3/4, and MUB5/6, suggesting func-tional diversification of plant MUBs. In contrast, multicellularfungi and animals have only a single MUB gene, and no MUBgene is evident in yeast (26). Although the structure and post-translational prenyl modifications of MUBs have been wellcharacterized, the function of these Ub-fold proteins isunknown.In this work, we demonstrate that AtMUB proteins interact

with a specific subgroup of E2s. We provide evidence thatMUBs directly interact with an E2 noncovalent binding surfacein vitro and validate the interaction in planta by co-immuno-precipitation (Co-IP) and bimolecular fluorescence comple-mentation (BiFC). From these data, we propose that MUB pro-teins contribute subcellular specificity to the ubiquitylationsystem by recruiting specific E2s to the plasma membrane.

EXPERIMENTAL PROCEDURES

Phylogenetic Sequence and Structure Analyses—E2 aminoacid sequences were trimmed to core domains as identifiedpreviously (27). Initial pairwise alignment of the sequences wasdone in ClustalX2 (28) using the PAM350 protein weightmatrix permitting gap opening and extension penalties of 35.0and 0.75. Final alignment (supplemental Fig. 1) was manuallyperformed with Se-Al Version 2.0a11 (29). The reported phy-logenetic tree is the strict consensus of three possible trees gen-erated by amaximumparsimony full heuristic search in PAUP*Version 4.0b10 (30) with AtUFC1 (E2 for UFM1) as an out-group. Bootstrap support was determined with 1000 pseu-doreplicates by the TBR branch swapping algorithm andmapped onto the strict consensus. Alignments were annotatedfor identity and conservation using Jalview Version 2.5 (31, 32).Protein structures for AtMUB1 (Protein Data Bank code 1SE9)and Ub-UbcH5c (code 2FUH) from the NCBIMolecular Mod-eling Structure Data Base were annotated using the PyMOLviewer.Vectors and Plasmid Construction—All GatewayTM entry

and ligation cloned vectors were verified by sequencing. Primersequences for entry vectors and mutagenesis are listed in sup-plemental Table I. For GatewayTM cloning (Invitrogen), geneswithin entry vectors were recombined into destination vectorsby LR Clonase II reactions and confirmed by restriction map-

ping. All Gateway cloned constructs are summarized in supple-mental Table II. Amino acid substitutions in AtMUB andAtUBC genes were made following the QuikChange site-directed mutagenesis protocol (Stratagene). Arabidopsis E2genes, excludingAtUBC7 andAtUBC23, were obtained fromtheArabidopsis Biological Resource Center (Columbus, OH)as cDNA clones in pDONR201 (27) (supplemental Table II).Construction of plasmids pDEST-GBKT7 (33); pACT2.2gtwy(Addgene plasmid 11346); pEarleyGate (pEG) 100, pEG104,pEG201, and pEG202 (34); pGGWA and pHGWA (35);pET28a-UbcH5c and pET28a-UbcH5c S22R (Addgene plas-mids 12643 and 12644) (12); and pET28b-AtMUB1–6 (26) wasdescribed previously. The pSAT1-CAMBIA-nEYFP-C1 andpSAT1-CAMBIA-cEYFP-C1 N-terminal split YFP BiFC trans-formation vectors were constructed with expression cassettesfrom pSAT1-nEYFP-C1 and pSAT1-cEYFP-C1 (36) insertedinto pCAMBIA0380 (37).To create Gateway entry vectors, AtMUB3 and AtMUB4

were PCR-amplified from published pET28 vectors (26) withforward primers including a 5�-CACC sequence and reverseprimers designating 3�-CAAX or 3�-SAAX; products weredirectionally cloned into pENTR/D-TOPO (Invitrogen).AtCOP10 and AtUEV1B were amplified from Columbia-0cDNA and HsMUB from SK-HEP cDNA (38) and similarlycloned into pENTR/D-TOPO. The AtCOP10 splice variantAt3g13550.1 was gel-excised prior to D-TOPO cloning. For theFLAG-tagged �-glucuronidase (GUS) control vector, PCR-modified pENTR-GUS (Invitrogen) was recombined intopEG202 (supplemental Table II).The mCerulean plant nuclear marker was created by PCR

amplifying the N-terminal nuclear localization signal (NLS) ofAtZFP11 (At2g42410) (39) from Columbia-0 genomic DNAwith primers adding a 5�-CACC and a 3�-XhoI site and insertedinto pENTR/D-TOPO. The mCerulean sequence was PCR-amplified from pmCerulean-C1 (40) to include a C-terminalstop codon (*) and flanking XhoI sites, ligated into similarlycut pENTR/D-TOPO�ZFP11(NLS), sequence-confirmed, andrecombined into pEG100 (34) to create the binary vectorpEG100�ZFP11(NLS):mCerulean*.Using conventional cloning methods, AtMUB1–3 and

AtMUB6 (CAAX and SAAX forms) and Ub were PCR-ampli-fied with flanking 5�-NdeI and 3�-PstI sites and cloned into thepGBKT7 yeast two-hybrid vector (Clontech). AtMUB4 andAtMUB5 were PCR-amplified with 5�-EcoRI and 3�-BamHIsites and cloned into pGBKT7. AtMUB3, AtMUB4, AtUBC8,and AtUBC9 were PCR-amplified from entry vectors with5�-XhoI and 3�-XbaI sites and cloned into similarly cut pSAT1-CAMBIA BiFC vectors.Yeast Two-hybrid Analyses—The yeast two-hybrid cDNA

library screen was performed by the Molecular InteractionFacility (Madison,WI) following standard protocols (41). Hap-loid yeast mating strains PJ694A (for MUB genes) and PJ694�(for UBC and AtUEV genes) (42) were transformed using theFrozen-EZ Yeast Transformation II method (Zymo ResearchCorp.). Yeast cells were grown at 30 °C on complete yeastextract peptone dextrose (YEPD) or synthetic dropoutmediumlacking amino acids as appropriate for mating and plasmidselection. Sterile 3-amino-1,2,4-triazole at 0.5, 1, 2, or 4 mM

Arabidopsis MUBs Localize E2s to the Plasma Membrane

14914 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 17 • APRIL 29, 2011

by guest on September 22, 2020

http://ww

w.jbc.org/

Dow

nloaded from

added stringency on synthetic dropout medium lacking histi-dine. Interaction data are reported only in the absence ofgrowth of empty vector controls. Results were confirmed withstandard �-gal filter lift assays (data not shown).Protein Purification and in Vitro Interaction Studies—Coex-

pression of AtMUB3 SAAX and AtUBC8 was carried out inEscherichia coli BL21. All other proteins were expressedin E. coli Rosetta 2 (Novagen). Expression of His6-taggedAtMUB3 was induced with 0.2 mM isopropyl 1-thio-�-D-galac-topyranoside for 12 h at 19 °C. Cells were extracted by ultra-sonication in PBS (20mM phosphate and 200mMNaCl, pH 6.8)and clarified by centrifugation. PMSF was maintained at 1 mM

throughout purification. Extract was incubated with TALONmetal affinity resin (Clontech) for 30 min, followed by threewashes with 10 mM imidazole/PBS, three washes with 50 mM

imidazole/PBS, and elution with 250 mM imidazole/PBS.Expression of GST-tagged AtUBC8 and AtUBC8(S22R) wasinduced as described above, and soluble protein extracts wereincubated with immobilized GSH resin (Thermo Scientific) for30 min, followed by three washes with PBS. GST-tagged pro-teins were eluted in 15 mM reduced GSH/PBS. For pulldownassays, GST-tagged AtUBC8 or AtUBC8(S22R) was immo-bilized on GSH resin, mixed with purified His6-taggedAtMUB3 for 30 min, washed three times with PBS, andboiled in Laemmli sample buffer. All purifications were ana-lyzed by SDS-PAGE and Coomassie Blue staining or immu-noblotting as appropriate.Agrobacterium Infiltration, Co-IP, and BiFC—Binary vectors

were transformed into Agrobacterium tumefaciens strainGV3101 by electroporation. Cells from an overnight culturewere washed with sterile 20 mM MgSO4 and suspended in anequal volume of infiltration buffer (10mMMES, 10mMMgSO4,and 100 �M acetosyringone, pH 5.2). Bacterial infiltration solu-tionswere incubated for 3 h at 25 °Cwith shaking beforemixingthe cultures as appropriate and infiltrating into young, nearlyexpanded Nicotiana benthamiana leaves. Plants (3–5 weeksold) grown in 16 h long days under fluorescent light were keptin the dark for 2 days following infiltration before beingreturned to the light for 1 day. Cells were imaged by confocalmicroscopy with a Zeiss LSM 5 microscope using an argonlaser. The YFP signal was obtained with 514 nm excitation withHFT 458/514, NFT 545, and BP 530–600 filters. For negativecontrols, cells within infiltrated regions of tissue wereimaged. Autofluorescence was excited at 514 nm and col-lected with HFT 458/514, NFT 545, and LP 650 filters, andmCerulean was excited at 458 nm and collected with HFT458/514/633, NFT 490, and BP 475–525 filters. Imagebrightness and contrast adjustments and overlays were donewith NIH ImageJ Version 1.44b.For Co-IP studies, Agrobacterium cultures were normalized

to A600 � 0.6 in infiltration buffer, mixed, and co-infiltrated.N. benthamiana tissue was ground with 3 volumes (w/v) ofprechilled extraction buffer (50mMTris adjusted to pH8.0withMES, 0.5 M sucrose, 1 mM MgCl2, 10 mM EDTA, and 5 mM

DTT) adapted from Liu et al. (43) with 1 mM PMSF and 1:100plant protease inhibitor mixture (Sigma) in a 4 °C cold room.The homogenate was centrifuged at 4000 � g for 15 min andclarified again at 2000 � g for 10 min. Total protein was quan-

tified by Coomassie Plus assay (Pierce), and samples were nor-malized to 2 mg/ml. The supernatant was incubated with anti-FLAGM2 affinity gel (Sigma) for 1 h at 4 °C, washed three timeswith PBS with 0.05% Tween 20, boiled with Laemmli samplebuffer, and analyzed by SDS-PAGE and immunoblotting withanti-HA-HRP and anti-FLAG antibodies (Sigma).

RESULTS

Yeast Two-hybrid Screen Reveals MUB-interacting E2s—AtMUB1 was used as bait in a Gal4 yeast two-hybrid (Y2H)Arabidopsis cDNA screen of �18 million clones representingmRNA from tissues, cultured cells, and diverse physiologicalconditions (Molecular Interaction Facility) and identified mul-tiple occurrences of three E2s, AtUBC8–10, with 20, 2, and 5hits, respectively. To facilitate nuclear interaction of theGal4 transcriptional subunits, a prenylation-deficient mutant,AtMUB1 SAAX (which corresponds to C114S), was used asbait. In directed Y2H experiments, bothWTCAAX and preny-lation-deficient SAAX forms of AtMUBs interacted withAtUBC8–10, but results using the SAAX versions were mostconsistent (data not shown). Therefore, MUB SAAX bait con-structs were used in subsequent experiments.ArabidopsisMUBs Specifically Interact with SubgroupVI E2s—

Directed Y2H experiments were performed to determinewhether MUBs interact with additional E2s that were unde-tected due to gene expression bias in the cDNA library and toestablish an interaction pattern between MUBs and E2s. Thir-ty-seven AtUBCs were organized into 14 subgroups by Kraft etal. (27) based on the phylogenetic relationship of the enzymaticcore domain. This diverseArabidopsis E2 family is representedin Fig. 1A with additional gene structure characteristics thatvalidate the well supported sequence relationships (bootstrapsupport in Fig. 1A). Haploid yeast strains for all six AtMUBSAAX and Ub baits were crossed with all 14 E2 subgroups asprey. In total, 35 of 37ArabidopsisE2s and twoUEVs,AtCOP10and AtUEV1B (MMZ2), were examined. AtUEV1B was testedbecause it is anArabidopsis homolog of Mms2 (17), and it con-tains a C-terminal CAAX (CCVM) signal that might localize itwith MUBs at a membrane. AtCOP10 was tested because,among the UEV family members, it is most closely related toAtUBC8–10 (Fig. 1A). In this comprehensive directed survey ofMUB-E2 interactions, all six AtMUBs interacted with E2s, con-firming the AtMUB1 library screen results (Fig. 1B).AtMUBs interacted exclusively with Subgroup VI E2s,

including AtUBC8–10, as detected in the initial Y2H screen,and also with AtUBC11 and AtUBC28–30 (Fig. 1B). AtUBC12and AtUBC19 failed to interact but were excluded from furtheranalysis because growth was auto-activated with various emptybait vector controls. Within the positive Subgroup VI interac-tions, MUBs showed a combinatorial pattern of interactionwith E2s. For instance, AtUBC9 interactedwith all sixAtMUBs,whereas AtUBC11 and AtUBC30 interacted only with theAtMUB3/4 subgroup (Fig. 1B). Ironically, the original bait,AtMUB1, showed the weakest E2 interaction profile, only pos-itive for AtUBC8 and AtUBC9 under the assay conditionsshown in Fig. 1B. No interaction was detected betweenAtMUBs and a SUMO E2, AtSCE1 (data not shown); between

Arabidopsis MUBs Localize E2s to the Plasma Membrane

APRIL 29, 2011 • VOLUME 286 • NUMBER 17 JOURNAL OF BIOLOGICAL CHEMISTRY 14915

by guest on September 22, 2020

http://ww

w.jbc.org/

Dow

nloaded from

AtMUBs and AtUEVs; or between Ub and any of the SubgroupVI E2s by Y2H survey.AtMUB3 and AtMUB4 yeast colonies showed robust

growth with the broadest number of E2s and were used forsubsequent experiments. AtMUB3 and AtMUB4 are repre-sentative of the two prenylation modifications: geranylgera-nylation and farnesylation, respectively (26). The AtMUB3/4subgroup also represents the residue variation seen in the UbIle44-homologous surface, where a number of UBDs areknown to interact (Fig. 2, C and D) (24). Mutation of theyeast nonessential T66 residue in Ub to E disrupts binding ofUBDs to the Ile44 surface while preserving Ub conjugationactivity (12, 44). Thr66 in Ub is substituted with Val inAtRUB and all known animal and fungal MUBs (26). In Ara-bidopsis, Val and Thr are alternately found at the same posi-tion in MUB3 and MUB4, respectively (Fig. 2C).Subgroup VI E2s and MUBs Contain Structure-predicted

Noncovalent Interaction Sites—We constructed an alignmentof MUB-interacting and MUB-non-interacting E2s to identifyany common sequence motif responsible for the Y2H interac-tion pattern. Fig. 2A illustrates sequence identity in a subregionof the E2 core encompassing�-sheets 1–3. As expected, there ishigh sequence conservation throughout the core, even whencomparing representative E2s of distantly related subgroups(supplemental Fig. 1). This relationship is evident in the num-ber of residues with 80% identity, highlighted in black in thealignment (Fig. 2A). However, the weak overall E2 conservationspanning �-sheets 1 and 2 contrasts with the strong conserva-tion within MUB-interacting E2s including an absolutely con-served Ser22, indicated in Fig. 2A.TheMUB-interacting Subgroup VI E2s are closely related to

the human UbcH5a–c and yeast Ubc4/5 E2 families. Non-covalent Ub contact residues identified on HsUbcH5b andHsUbcH5c span �-sheets 1–3 and are also conserved in MUB-interacting E2s (Fig. 2, A and B). We found that HsMUB also

interacted with HsUbcH5c by Y2H assay (Fig. 2E). In fact,HsMUB interacted with AtUBC8 and AtUBC9, and AtMUB3and AtMUB4 interacted with HsUbcH5c, demonstrating anevolutionarily conserved interface.Conserved sequence between Arabidopsis MUBs and Ub is

depicted in Fig. 2C. Ub-E2 contact residues conserved inMUBsform a discrete Ub Ile44-proximal surface including Thr66 (Fig.2D) and, in conjunction with E2 conservation, led to thehypothesis that a similar noncovalent interaction occursbetween MUBs and Subgroup VI E2s.MUBs Interact Noncovalently with E2s on a Surface Distinct

from the E2 Active Site—To determine whether MUBs and E2sinteract in a fashion analogous to Ub and HsUbcH5b/c,mutagenesis was performed in the predicted interaction sitesbased on the mutations HsUbcH5c (S22R) and Ub (T66E) (12).HomologousV86E andT86Emutationsweremade inAtMUB3and AtMUB4, respectively. Likewise, AtUBC8 (S22R) andAtUBC9 (S52R) noncovalent interaction site mutations andactive site C85S and C115S mutations were constructed. Asshown in Fig. 3, the S22R and S52R mutations in AtUBC8 andAtUBC9 completely abolished MUB-E2 interaction by Y2Hassay. The homologous HsUbcH5c (S22R) mutation also inter-rupted the interactionwithHsMUB (supplemental Fig. 3). Sim-ilarly, the noncovalent interaction mutants AtMUB3 (V86E)andAtMUB4 (T86E) were unable to interact with eitherWTormutant E2s. Alternatively, E2 active site mutations did notinterfere with MUB interaction by Y2H assay. Taken togetherwith the strong sequence and structural conservation, thesedata support the conclusion that MUBs and Ub interact withthis versatile E2 interface.MUBs and E2s Bind Directly in Vitro—To establish whether

MUB-E2 binding is dependent upon additional eukaryotic pro-teins, the interaction was reconstituted in vitro. Recombinantpurified AtMUB3was pulled down byGST-AtUBC8 but not byGST-UBC8 (S22R) or GST alone (Fig. 4A), demonstrating that

FIGURE 1. Arabidopsis MUBs specifically interact with Subgroup VI E2s. A, a strict consensus phylogenetic tree summarizes the sequence relationshipwithin the core domain of Arabidopsis E2s. Subgroups are indicated with Roman numerals, as described (23). The tree is rooted to outgroup AtUFC1, the E2 forUFM conjugation. Asterisks indicate branches with 100% bootstrap support from 1000 pseudoreplicates. Additional E2 gene characters, including the lengthof the N-terminal extension (N), core domain (Cr), and C-terminal extension (C) and the average number of introns (I), are shaded according to the key (inset).B, a directed Y2H assay of positive interactions identified in a screen between the baits AtMUB1– 6 and Ub and preys AtUBC1– 6, AtUBC8 –22, AtUBC24 –37,AtCOP10, and AtUEV1B. The entire screen and full-size tree are available in supplemental Fig. 2. DNA-binding domain bait constructs and corresponding vectorcontrols (V) are on the horizontal axis, whereas activation domain prey constructs and vector controls are on the vertical axis. Y2H selection is summarized foreach panel (lower), where amino acid dropouts (�) are indicated: L, leucine; W, tryptophan; H, histidine; 3-AT, 2 mM 3-amino-1,2,4-triazole.

Arabidopsis MUBs Localize E2s to the Plasma Membrane

14916 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 17 • APRIL 29, 2011

by guest on September 22, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Ser22 is required for direct binding to AtMUB3. We wereunable to perform the reciprocal experiment because purifiedAtMUB3 was unstable when reapplied to metal affinity resin.We circumvented this detail by using lysates from E. coli coex-pressing the proteins. Here, AtUBC8 pulled down AtMUB3and vice versa, but AtUBC8 (S22R) only weakly boundAtMUB3 (Fig. 4B). These in vitro assays confirm the Y2H inter-

action data and also establish that MUB-E2 binding can occurin the absence of additional eukaryotic proteins.MUB and E2 Noncovalent Interaction Mutations Prevent

Co-IP in Planta—To examine the MUB-E2 interface in planta,anti-FLAGCo-IPwas performed onN. benthamiana leaf tissueco-infiltrated with combinations of WT or mutant FLAG-At-MUB3 CAAX and HA-AtUBC8 constructs using FLAG-GUS

FIGURE 2. Arabidopsis Subgroup VI E2s and MUBs contain structure-predicted noncovalent interaction sites. A, protein sequence alignment of �-helix 1through �-sheet 4 of MUB-interacting and representative non-MUB-interacting E2s (identified in Fig. 1) is indicated on the vertical axis. Sequence identities of80% (black) and 50% (gray) are highlighted. Also included are human E2s previously determined to bind Ub in this region, including HsUbcH5c (12) andHsUbcH5b (14). Residues NMR-shifted by Ub noncovalent binding with the HsUbcH5 family are indicated: closed circles and triangles, HsUbcH5b and HsUbcH5c;open circles, UbcH5b or UbcH5c. The interaction-critical HsUbcH5 Ser22 is indicated by a triangle. The homologous yeast Saccharomyces cerevisiae (Sc) Ubc4 isincluded for reference. B, a surface-rendered HsUbcH5c structure (Protein Data Bank code 2FUH) shows the noncovalent Ub-binding interface with the Cterminus indicated (asterisk). Residues indicated with circles or a triangle in A were colored onto the structure if conserved in MUB-interacting E2s: orange, closedcircles (HsUbcH5 Pro16, Val26, Gln34, Met38, and Val49); yellow, open circles (HsUbcH5 Pro17, Ala23, Thr36, and Gly47); red, triangle (HsUbcH5 Ser22). C, proteinsequence alignment of AtMUBs and Ub. A sequence identity of 80% (black) and a conservation index of 50% (gray) are highlighted. D, residues indicated in Cwith circles or a triangle were colored onto the structures if conserved between AtMUBs and Ub: teal, closed circles (Ub Lys6, Arg42, Ile44, Lys48, Thr66, and Val70);white, open circles (Ub Gly10, Ala46, Gly47, and His68); blue, triangle (Ub Thr66, AtMUB1 Thr87, AtMUB3 Val86, AtMUB4 Thr86). E, a directed Y2H assay demonstratingthe interaction of HsUbcH5c with HsMUB, as well as the cross-kingdom interactions of AtMUBs with HsUbcH5c and of HsMUB with AtUBCs. Assay conditionswere as described in the legend to Fig. 1, except that 1 mM 3-amino-1,2,4-triazole (3-AT) was used. V, vector control.

Arabidopsis MUBs Localize E2s to the Plasma Membrane

APRIL 29, 2011 • VOLUME 286 • NUMBER 17 JOURNAL OF BIOLOGICAL CHEMISTRY 14917

by guest on September 22, 2020

http://ww

w.jbc.org/

Dow

nloaded from

as a negative control. To compensate for lower AtUBC8 (S22R)expression, three times more total extract was loaded (Fig. 5A,fourth and fifth lanes) and applied to resin relative to WTAtUBC8. WT AtUBC8 coprecipitated only with WT AtMUB3but not with AtMUB3 (V86E) or GUS. AtUBC8 (S22R) failed tocoprecipitate with either WT or mutant AtMUB3 (Fig. 5A).MUBs Localize E2s to the PlasmaMembrane in Planta—We

additionally sought to identify the localization of the MUB-E2interaction through BiFC of split YFP. A full-length YFP fusionof AtMUB3 CAAX localized to the plasma membrane inN. benthamiana leaf cells (Fig. 5B). Alternately, YFP-taggedAtMUB3 SAAX, AtUBC8, and AtUBC9 all localized in nucleiand the cytoplasm. These MUB localization results in N. ben-thamiana are similar to previous observations in Arabidopsisprotoplasts and stably transformed plants (26).When N-terminal YFP-AtMUB3 SAAX was co-infiltrated

with C-terminal YFP-AtUBC8 or C-terminal YFP-AtUBC9,

fluorescence was observed in nuclei and cytoplasmicstrands, indicating that the proteins do indeed interact (Fig.5C). Importantly, when N-terminal YFP-AtMUB3 CAAXwas co-infiltrated with C-terminal YFP-AtUBC8, not onlywas fluorescence observed, but it was localized to the plasmamembrane (Fig. 5, C and D). The same result was seen withAtMUB3 CAAX and AtUBC9. Likewise, AtMUB4 CAAX co-infiltration with AtUBC8 or AtUBC9 also localized fluores-cence to the plasma membrane (supplemental Fig. 4). SplitYFP fusions of MUBs or E2s did not fluoresce when coex-pressed with the complementing split YFP empty vector (Fig.5C, left and top panels). The BiFC experiments demon-strated that transgenic MUB fusions are properly recognizedby prenylation machinery in plants and, as predicted, local-ized to the plasma membrane. These experiments alsoshowed that MUBs and E2s interact in vivo and that MUBprenylation and membrane localization are sufficient to, at aminimum, recruit E2s to the plasma membrane.

DISCUSSION

Prior to thiswork, the family of eukaryoticMUBproteinswasdetermined to be associated with the plasmamembrane in vivobut remained functionally uncharacterized. Here, we have pro-vided the first evidence that MUBs are a direct physical linkbetween the plasma membrane and the ubiquitylation system.Specifically, MUBs recruit a subset of E2s that are defined by astructurally conserved interface, which also interacts noncova-lently with Ub (12–14, 45) and possibly other Ub-like proteins.Y2H, pulldown, and in plantaCo-IP assays with noncovalent

surface mutations identified the interface for the MUB-E2interaction. Co-IPs performed usingMUBCAAX proteins par-alleled the native localization of MUBs with plasma membraneperipheral proteins. Furthermore, BiFC experiments con-firmed the interaction and demonstrated that prenylatedMUBproteins can simultaneously bind to the plasmamembrane and

FIGURE 3. Arabidopsis MUBs interact with E2s on a surface distinct fromthe E2 active site. Shown are the results from a Y2H mutation analysis of theUb-HsUbcH5 equivalent noncovalent interaction surfaces of AtMUBs (MUB3V86E and MUB4 T86E) and AtUBCs (UBC8 S22R and UBC9 S52R) or AtUBCactive sites (UBC8 C85S and UBC9 C115S) as described in the legend to Fig. 1,except that 4 mM 3-amino-1,2,4-triazole (3-AT) was used. V, vector control.

FIGURE 4. Arabidopsis MUBs interact directly with E2s in vitro. A, a GST pulldown assay using purified proteins reconstituted MUB-E2 binding in vitro. Inputsof recombinant GST-AtUBC8, GST-AtUBC8 S22R, or GST alone were incubated with His-AtMUB3. GSH resin eluates were examined by SDS-PAGE and immu-noblotting as indicated. Notably, the interaction was sensitive to the S22R mutation. B, E. coli coexpression lysates were purified with either GSH resin or TALONHis affinity (metal) resin as indicated. Coexpression cultures were achieved with double antibiotic selection for His-AtMUB3 (Ampr) and GST-AtUBC8 orGST-AtUBC8 S22R (Kanr). GST-AtUBC8 and His-AtMUB3 proteins copurified efficiently. GST-AtUBC8 S22R and AtMUB3 failed to copurify effectively, as detectedby Coomassie Blue, despite similar input protein levels in total E. coli lysates detected by immunoblotting as indicated. AtUBC8 and AtMUB3 CoomassieBlue-stained bands are identified with arrows. Substoichiometric Coomassie Blue-stained proteins migrating between 21.5 and 31 kDa were recovered witheither purification as resin-binding background.

Arabidopsis MUBs Localize E2s to the Plasma Membrane

14918 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 17 • APRIL 29, 2011

by guest on September 22, 2020

http://ww

w.jbc.org/

Dow

nloaded from

to E2s. On the basis of this, we hypothesize that MUBs recruitE2s, perhaps to regulate the homeostasis of active Ub in theplasma membrane-proximal space.The seven Arabidopsis Subgroup VI E2s that interact with

MUBs are essentially composed of the core domain, are activewith a wide array of E3s (27, 46), and have been characterizedextensively in vitro (47, 48). Related E2s in animals and fungi,including the human UbcH5 and yeast Ubc4/5 families, havealso served as robust model E2s. It is possible that the small sizeof the Subgroup VI E2s permits access to a diverse array oftargets while restricted to the two-dimensional cytoplasmicsurface of the plasma membrane. Here, by identifying the

capacity to be recruited to the plasma membrane by MUBproteins, we have added a distinguishing biological trait forthis E2 family.We were unable to detect MUB Y2H interactions with E2s

outside of this subgroup, including the UEVs AtCOP10 andAtUEV1B (Fig. 1A and supplemental Fig. 2). Published NMRstudies corroborate thatAtCOP10, although sister to theMUB-interacting subgroup, does not interact noncovalently with Ub(13, 15).The Mms2-related AtUEV1B and the HsUbcH5-related

Arabidopsis Subgroup VI E2s also did not interact with Ub byY2H assay. This result is not contradictory because the initial

FIGURE 5. Arabidopsis MUBs localize E2s to the plasma membrane in planta. A, a Co-IP assay with anti-FLAG resin on N. benthamiana leaf tissue cotrans-formed with WT or mutant combinations of HA-AtUBC8 and FLAG-AtMUB3 CAAX or the FLAG-GUS control as indicated. AtUBC8 coprecipitated with AtMUB3but not with AtMUB3 V86E or GUS. AtUBC8 S22R did not coprecipitate with either AtMUB3 or AtMUB3 V86E. For AtUBC8-containing extracts, 16 �g of totalprotein was loaded on SDS-polyacrylamide gel representing input, and 450 �g of total protein was applied to 40 �l of anti-FLAG resin. To analyze similaramounts of UBC, three times more AtUBC8 S22R-containing extract was loaded on SDS-polyacrylamide gel and applied to 40 �l of anti-FLAG resin. Equalvolumes of FLAG Co-IP eluates were analyzed by SDS-PAGE and immunoblotting as indicated. The asterisk indicates mouse IgG cross-reacting bands. B, N. ben-thamiana epidermal cells transiently expressing full-length YFP fusions of plasma membrane localized AtMUB3 CAAX or cytoplasmic/nuclear localized AtMUB3SAAX, AtUBC8, and AtUBC9. YFP signals localized to cytoplasmic strands (closed arrowheads) and nuclei (open arrowheads) are indicated. C, BiFC of epidermalcells coexpressing split YFP fusions of AtMUB3 and AtUBCs or empty vector controls. Combinations of N- and C-terminal YFP fragments (nY and cY, respectively)were infiltrated as vector controls or fused to the N terminus of AtMUB and E2 as indicated. AtMUB-E2 interaction and co-localization were observed at theplasma membrane or in the cytoplasm (closed arrowheads) and nucleus (open arrowheads). D, an enlarged view of BiFC with N-terminal YFP-AtMUB3 CAAX orN-terminal YFP-AtMUB3 SAAX and C-terminal YFP-AtUBC8 highlighting fluorescence in cytoplasmic strands (closed arrowheads) and the nucleus (open arrow-head). Also shown is chloroplast autofluorescence (red) and nuclear localized AtZFP11(NLS)-Cerulean (blue). Overlap between YFP and Cerulean is indicated(white) in the merged images. Scale bars � 10 �m. E, a model depicting prenylation-dependent membrane-localized MUB (prenylcysteine (C)) tethering an E2to the inner surface of the plasma membrane (PM), leaving the E2 active site (cysteine (C)) available to bind the diglycine (GG) of Ub. Interaction-critical residuesbetween MUB (T/V) and E2 (S) are highlighted. Various proteins that could join the complex are indicated in gray: Ub, E3, target proteins (T), and available lysines (K).

Arabidopsis MUBs Localize E2s to the Plasma Membrane

APRIL 29, 2011 • VOLUME 286 • NUMBER 17 JOURNAL OF BIOLOGICAL CHEMISTRY 14919

by guest on September 22, 2020

http://ww

w.jbc.org/

Dow

nloaded from

evaluations of Mms2 and HsUbcH5 with Ub were performedwith sensitive NMR-based methods (12, 14, 16). However,this does raise the possibility that MUBs bind Subgroup VIE2s with greater affinity than the Ub-E2 interactionsdescribed previously.Individual subgroup VI E2s likely serve specialized functions

in planta, as suggested by the combinatorial pattern of Y2Hinteractions with MUBs. For instance, the AtUBC29 andAtUBC30 proteins are 95% similar with comparable expressionin planta (27), but only AtUBC29 interacts with AtMUB6. Thispattern echoes previously described combinatorial UPS inter-actions among SCF (for Skp1, Cullin, F-box) E3 subunits (49) orbetween an E3 andmultiple E2s (46). Themodular AtMUB andE2 interactions are consistent with an expanded plant UPScompared with animals and fungi (46, 48, 49). Regardless of thespecies, MUBs may help refine ubiquitylation target selection,reaction dynamics, or localization.MUB prenylation and E2 binding ability are reminiscent of

Pex22p and Cue1p localizing E2s to the endoplasmic reticulumand peroxisome membranes, respectively. In yeast, the trans-membrane protein Pex22p recruits the E2 Pex4p to the outerperoxisomal membrane, causing monoubiquitylation andexport of Pex5p to the cytoplasm (50, 51). This process appearsto be conserved in plants, as a functionally equivalent AtPEX4-AtPEX22 complex is needed to sustain peroxisome biogenesis(52, 53). Also in yeast, the transmembrane protein Cue1precruits the E2 Ubc7p to the cytoplasmic surface of the endo-plasmic reticulum for endoplasmic reticulum-associated deg-radation of errant proteins (54–57). Now, with the addition ofMUBs, a theme emerges in which small adapter proteinsanchor E2s to the cytoplasmic surface of specific membranes.Future studies will determine whether MUBs also mediate Ub-directed movement of membrane proteins into the cytoplasmlike Pex22p and Cue1p.In the simplest model,MUBs could recruit an activated E2 to

the plasma membrane to donate its Ub to a target or an elon-gating Ub chain (Fig. 5E). Alternatively, the recruited E2 coulddonate its Ub to Ub-equivalent Lys29, Lys48, or Lys63 (26) on aMUB protein. Ubiquitylation of these lysines would presentmono-Ub for UBD docking or establish membrane-localizedreservoirs of various Ub chains.MUBsmay also influence the dynamics of polyubiquitylating

complexes. Recent studies have identified that noncovalent Ubinteractions with E2s are important for regulating Ub chainformation (12–14, 19, 45). HsUbcH5c noncovalent interactionwith Ub and the subsequent formation of high molecularweight (Ub�E2)n polymers are required for auto-polyubiquity-lation of the E3 BRCA1 (12). Here, the S22R mutant ofHsUbcH5c is unable to support (Ub�E2)n polymer formationbut can still sustain auto-monoubiquitylation of BRCA1 in vitro(12–14, 45). The shared use of the E2 noncovalent interactionsurface byMUB andUb poses several possibilities whereMUBsinfluence Ub chain formation (Fig. 5E). MUB competition withUb for noncovalent E2 binding could promote monoubiquity-lation ofmembrane substrates by causing a localized disruptionin (Ub�E2)n polymer formation. In contrast, MUBs could takethe place of a terminal Ub in a (Ub�E2)n polymer to facilitatethe rapid assembly of poly-Ub chains on a membrane target.

Furthermore, the binding ofMUB toE2s could affect E2 activityregarding chain linkage specificity (58), enzymatic rate (15, 27),and E3 selection (27, 46). Although the impact of MUB-E2binding at the plasma membrane is uncertain, these modelshighlight the potential for key advances in understandingubiquitylation at the plasma membrane and warrant furtherinvestigation.

Acknowledgments—Protein expression vectors pGGWAandpHGWAwere generous gifts from Didier Busso. pACT2.2gtwy was created byGuy Caldwell. BiFC binary plasmids pSAT1-CAMBIA-nEYFP-C1and pSAT1-CAMBIA-cEYFP-C1 were gifts from Sona Pandey. ThepmCerulean-C1 plasmid template was a gift from Howard Berg withthe permission of David Piston. Human SK-HEP cDNA for HsMUBwas a gift from Barrie Bode and Richard Finger. We appreciate thephylogenetic help from Mauricio Diazgranados and Jan Barber; theBiFC advice from Naveen Bisht; the preliminary BiFC assistanceusing Physcomitrella patens from Julie Thole, Pierre-Francois Per-roud, and Ralph Quatrano; and the use of an ImageQuant LAS 3000imager from John Chrivia.

REFERENCES1. Vierstra, R. D. (2009) Nat. Rev. Mol. Cell Biol. 10, 385–3972. Komander, D. (2009) Biochem. Soc. Trans. 37, 937–9533. Woelk, T., Sigismund, S., Penengo, L., and Polo, S. (2007) Cell Div. 2, 114. Hicke, L., Schubert, H. L., and Hill, C. P. (2005)Nat. Rev. Mol. Cell Biol. 6,

610–6215. Dupre, S., Urban-Grimal, D., and Haguenauer-Tsapis, R. (2004) Biochim.

Biophys. Acta 1695, 89–1116. Hicke, L., and Dunn, R. (2003) Annu. Rev. Cell Dev. Biol. 19, 141–1727. Downes, B., and Vierstra, R. D. (2005) Biochem. Soc. Trans. 33, 393–3998. Farmer, L. M., Book, A. J., Lee, K. H., Lin, Y. L., Fu, H., and Vierstra, R. D.

(2010) Plant Cell 22, 124–1429. Geng, J., and Klionsky, D. J. (2008) EMBO Rep. 9, 859–86410. Lee, I., and Schindelin, H. (2008) Cell 134, 268–27811. Fu, H., Lin, Y. L., and Fatimababy, A. S. (2010) Trends Plant Sci. 15,

375–38612. Brzovic, P. S., Lissounov, A., Christensen, D. E., Hoyt, D. W., and Klevit,

R. E. (2006)Mol. Cell 21, 873–88013. Brzovic, P. S., and Klevit, R. E. (2006) Cell Cycle 5, 2867–287314. Sakata, E., Satoh, T., Yamamoto, S., Yamaguchi, Y., Yagi-Utsumi, M., Ku-

rimoto, E., Tanaka, K., Wakatsuki, S., and Kato, K. (2010) Structure 18,138–147

15. Lau, O. S., and Deng, X. W. (2009) Biochem. J. 418, 683–69016. Lewis, M. J., Saltibus, L. F., Hau, D. D., Xiao, W., and Spyracopoulos, L.

(2006) J. Biomol. NMR 34, 89–10017. Wen, R., Torres-Acosta, J. A., Pastushok, L., Lai, X., Pelzer, L., Wang, H.,

and Xiao, W. (2008) Plant Cell 20, 213–22718. Duda, D. M., van Waardenburg, R. C., Borg, L. A., McGarity, S., Nourse,

A.,Waddell,M. B., Bjornsti,M. A., and Schulman, B. A. (2007) J. Mol. Biol.369, 619–630

19. Knipscheer, P., van Dijk, W. J., Olsen, J. V., Mann, M., and Sixma, T. K.(2007) EMBO J. 26, 2797–2807

20. Bencsath, K. P., Podgorski,M. S., Pagala, V. R., Slaughter, C. A., and Schul-man, B. A. (2002) J. Biol. Chem. 277, 47938–47945

21. Capili, A. D., and Lima, C. D. (2007) J. Mol. Biol. 369, 608–61822. Liu, Q., Jin, C., Liao, X., Shen, Z., Chen, D. J., and Chen, Y. (1999) J. Biol.

Chem. 274, 16979–1698723. Tatham, M. H., Kim, S., Yu, B., Jaffray, E., Song, J., Zheng, J., Rodriguez,

M. S., Hay, R. T., and Chen, Y. (2003) Biochemistry 42, 9959–996924. de la Cruz, N. B., Peterson, F. C., Lytle, B. L., and Volkman, B. F. (2007)

Protein Sci. 16, 1479–148425. Vinarov, D. A., Lytle, B. L., Peterson, F. C., Tyler, E.M., Volkman, B. F., and

Markley, J. L. (2004) Nat. Methods 1, 149–153

Arabidopsis MUBs Localize E2s to the Plasma Membrane

14920 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 17 • APRIL 29, 2011

by guest on September 22, 2020

http://ww

w.jbc.org/

Dow

nloaded from

26. Downes, B. P., Saracco, S. A., Lee, S. S., Crowell, D. N., and Vierstra, R. D.(2006) J. Biol. Chem. 281, 27145–27157

27. Kraft, E., Stone, S. L., Ma, L., Su, N., Gao, Y., Lau, O. S., Deng, X. W., andCallis, J. (2005) Plant Physiol. 139, 1597–1611

28. Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan,P. A., McWilliam, H., Valentin, F., Wallace, I. M., Wilm, A., Lopez, R.,Thompson, J. D., Gibson, T. J., and Higgins, D. G. (2007) Bioinformatics23, 2947–2948

29. Rambaut, A. (1996–2002) Sequence Alignment (Se-Al) Program, Version2.0a11, University of Oxford, Oxford

30. Swofford, D. L. (2003) PAUP*, Phylogenetic Analysis Using Parsimony(and Other Methods), Version 4.0b10, Sinauer Associates Inc. Publishers,Sunderland, MA

31. Waterhouse, A. M., Procter, J. B., Martin, D. M., Clamp, M., and Barton,G. J. (2009) Bioinformatics 25, 1189–1191

32. Livingstone, C. D., and Barton, G. J. (1993) Comput. Appl. Biosci. 9,745–756

33. Rossignol, P., Collier, S., Bush,M., Shaw, P., andDoonan, J. H. (2007) J. CellSci. 120, 3678–3687

34. Earley, K. W., Haag, J. R., Pontes, O., Opper, K., Juehne, T., Song, K., andPikaard, C. S. (2006) Plant J. 45, 616–629

35. Busso, D., Delagoutte-Busso, B., andMoras, D. (2005)Anal. Biochem. 343,313–321

36. Citovsky, V., Lee, L. Y., Vyas, S., Glick, E., Chen, M. H., Vainstein, A.,Gafni, Y., Gelvin, S. B., and Tzfira, T. (2006) J. Mol. Biol. 362, 1120–1131

37. Hajdukiewicz, P., Svab, Z., and Maliga, P. (1994) Plant Mol. Biol. 25,989–994

38. Fuchs, B. C., Finger, R. E., Onan, M. C., and Bode, B. P. (2007) Am. J.Physiol. Cell Physiol. 293, C55–C63

39. Dinkins, R. D., Pflipsen, C., and Collins, G. B. (2003) Plant Sci. 165, 33–4140. Rizzo, M. A., Springer, G. H., Granada, B., and Piston, D. W. (2004) Nat.

Biotechnol. 22, 445–44941. Fields, S., and Song, O. (1989) Nature 340, 245–24642. James, P., Halladay, J., and Craig, E. A. (1996) Genetics 144, 1425–143643. Liu, L., Zhang, Y., Tang, S., Zhao, Q., Zhang, Z., Zhang, H., Dong, L., Guo,

H., and Xie, Q. (2010) Plant J. 61, 893–90344. Sloper-Mould, K. E., Jemc, J. C., Pickart, C.M., and Hicke, L. (2001) J. Biol.

Chem. 276, 30483–3048945. Christensen, D. E., Brzovic, P. S., and Klevit, R. E. (2007) Nat. Struct. Mol.

Biol. 14, 941–94846. Stone, S. L., Hauksdottir, H., Troy, A., Herschleb, J., Kraft, E., and Callis, J.

(2005) Plant Physiol. 137, 13–3047. Girod, P. A., and Vierstra, R. D. (1993) J. Biol. Chem. 268, 955–96048. Smalle, J., and Vierstra, R. D. (2004) Annu. Rev. Plant Biol. 55, 555–59049. Gagne, J. M., Downes, B. P., Shiu, S. H., Durski, A. M., and Vierstra, R. D.

(2002) Proc. Natl. Acad. Sci. U.S.A. 99, 11519–1152450. Platta, H. W., and Erdmann, R. (2007) Trends Cell Biol. 17, 474–48451. Kragt, A., Voorn-Brouwer, T., van den Berg, M., and Distel, B. (2005)

J. Biol. Chem. 280, 7867–787452. Khan, B. R., and Zolman, B. K. (2010) Plant Physiol. 154, 1602–161553. Zolman, B. K., Monroe-Augustus, M., Silva, I. D., and Bartel, B. (2005)

Plant Cell 17, 3422–343554. Kostova, Z., Mariano, J., Scholz, S., Koenig, C., and Weissman, A. M.

(2009) J. Cell Sci. 122, 1374–138155. Biederer, T., Volkwein, C., and Sommer, T. (1997) Science 278,

1806–180956. Ravid, T., and Hochstrasser, M. (2007) Nat. Cell Biol. 9, 422–42757. Koller, A., Snyder, W. B., Faber, K. N., Wenzel, T. J., Rangell, L., Keller,

G. A., and Subramani, S. (1999) J. Cell Biol. 146, 99–11258. David, Y., Ziv, T., Admon, A., and Navon, A. (2010) J. Biol. Chem. 285,

8595–8604

Arabidopsis MUBs Localize E2s to the Plasma Membrane

APRIL 29, 2011 • VOLUME 286 • NUMBER 17 JOURNAL OF BIOLOGICAL CHEMISTRY 14921

by guest on September 22, 2020

http://ww

w.jbc.org/

Dow

nloaded from

DownesRebecca T. Dowil, Xiaolong Lu, Scott A. Saracco, Richard D. Vierstra and Brian P.

Specific Subset of Ubiquitin-conjugating (E2) Enzymes to the Plasma Membrane Membrane-anchored Ubiquitin-fold (MUB) Proteins Localize aArabidopsis

doi: 10.1074/jbc.M110.158808 originally published online February 23, 20112011, 286:14913-14921.J. Biol. Chem. 

  10.1074/jbc.M110.158808Access the most updated version of this article at doi:

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

Supplemental material:

  http://www.jbc.org/content/suppl/2011/02/23/M110.158808.DC1

  http://www.jbc.org/content/286/17/14913.full.html#ref-list-1

This article cites 56 references, 19 of which can be accessed free at

by guest on September 22, 2020

http://ww

w.jbc.org/

Dow

nloaded from